期刊
JOURNAL OF THE AMERICAN HEART ASSOCIATION
卷 2, 期 4, 页码 -出版社
WILEY
DOI: 10.1161/JAHA.113.000318
关键词
CaMKII; mass spectrometry; phosphorylation; proteomics; transgenic mouse model
资金
- Fondation Leducq: the Alliance for CaMKII Signaling in Heart Disease
- Netherlands Proteomics Centre in the Netherlands Genomics Initiative
- NIH [R01HL70250, R01HL079031, R01HL113001, R01HL096652]
- Utrecht University's Focus Massa program
Background-The multifunctional Ca2+- and calmodulin-dependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful approach to determining CaMKII's role therein is large-scale analysis of phosphorylation events by mass spectrometry. However, current large-scale phosphoproteomics approaches have proved inadequate for high-fidelity identification of kinase-specific roles. The purpose of this study was to develop a phosphoproteomics approach to specifically identify CaMKII's downstream effects in cardiac tissue. Methods and Results-To identify putative downstream CaMKII targets in cardiac tissue, animals with myocardial-delimited expression of the specific peptide inhibitor of CaMKII (AC3-I) or an inactive control (AC3-C) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce CaMKII activation downstream of beta-adrenergic receptor agonist stimulation. Enriched phosphopeptides from AC3-I and AC3-C mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and high-resolution LC-MS/MS. Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC3-I-mediated CaMKII inhibition. Conclusions-Our data set included known CaMKII substrates, as well as several new candidate proteins involved in functions not previously implicated in CaMKII signaling.
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